Methods for amplifying a target nucleic acid sequence that may be present in a test sample are, by now, well known in the art. Such methods include the polymerase chain reaction (PCR) and ligase chain reaction (LCR). These methods have found widespread application in the medical diagnostic field as well as the fields of genetics, molecular biology and biochemistry.
In PCR, a pair of primers are employed in excess to hybridize at the outside ends of complementary strands of the target nucleic acid. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand. New primers are then hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules. PCR is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202.
LCR is an alternative mechanism for target amplification. In LCR, two sense (first and second) probes and two antisense (third and fourth) probes are employed in excess over the target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being positioned adjacent to each other so that the primary probes can be ligated into a fused product. Further, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar ligatable fashion. If the target is initially double stranded, the secondary probes will also hybridize to the target complement in the first instance. Once the fused strand of sense and antisense probes are separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary fused product. The fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described in EP-A-320,308, hereby incorporated by reference. Other aspects of LCR technique such as gap LCR (GLCR) are disclosed in EP-A-439,182, to K. C. Backman et al., hereby incorporated by reference.
Unfortunately, one drawback of nucleic acid amplification reactions is that they are mostly qualitative. The nature of amplification reactions makes it difficult for them to be used to quantitatively detect the presence of a target sequence which may be present in a test sample. Accordingly, while traditional amplification reactions are useful for detecting the presence of a minute quantity of a target sequence in a test sample, traditional amplification reactions generally cannot be employed to determine the quantity of a target sequence in a test sample.
Some traditional amplification reactions have been modified to enable quantitative amplification reaction analysis. One such quantitative amplification reaction is called "competitive amplification." This method is commonly applied to PCR. According to this method, a standard nucleic acid sequence competes with a target sequence during the amplification reaction. Generally, the standard sequence and a sample suspected of containing a target sequence are combined in a dilution series in which the amount of the standard sequence is constant in all members of the series. Alternatively, the standard sequence and sample sequence are combined in a dilution series in which the amount of standard sequence is varied among the members of the dilution series. In any case, the concentration of the standard sequence in the members of the dilution series is known. PCR is then performed on all members of the dilution series and results in the production of a mixture of two nucleic acid species. One species is derived from the standard sequence and one species is derived from the sample sequence. The concentration of each species in a particular dilution depends on the number of copies of the standard and the sample sequences in the dilution prior to amplification. During the amplification reaction, detectable groups are typically introduced into both types of sequences. After amplification, the two species are separated and the amount of detectable group incoporated into each species is determined. This detection procedure is performed for each member of the dilution series. A competition curve can then be generated and the amount of sample sequence can be extrapolated based on the known amounts of standard sequence.
Methods of competitive amplification have been described in U.S. Pat. No. 5,219,727; Jalava et al., BioTechniques 15:134-39 (1993). While these competitive amplification techniques have shown utility, they require substantial amounts of sample preparation time as well as technician interaction and concomitant risk of sample contamination. In addition, performing amplification on members of a dilution series requires more reagents than performing amplification on a single sample. All of these factors add to the costs of performing competitive amplification reactions.
Moreover, all prior art methods involve PCR amplification wherein the middle part of the standard sequence is completely unrelated to the target sequence to be detected, and is flanked by target-specific primers to used to differentiate the standard from the target sequence. Thus, the two sequences are easily differentiated. Such a method is incompatible with LCR amplification because the middle part of both the target and standard sequences is critical to the success of the amplification reaction. No prior art method existed, prior to Applicant's invention, for differentiating a standard sequence from a target sequence using LCR amplification.
Thus, there is a need for a method of quantitatively performing an amplification reaction, wherein the middle part of the standard sequence is substantially similar to the target sequence, which does not require amplifying a series of concentration standards alongside each assay to be quantitated, and can be employed in a clinical laboratory setting.